The African-centric P47S Variant of TP53 Confers Immune Dysregulation and Impaired Response to Immune Checkpoint Inhibition

The tumor suppressor TP53 is the most frequently mutated gene in cancer and is mutationally inactivated in 50% of sporadic tumors. Inactivating mutations in TP53 also occur in Li Fraumeni syndrome (LFS). In addition to germline mutations in TP53 in LFS that completely inactivate this protein, there are many more germline mutant forms of TP53 in human populations that partially inactivate this protein: we call these partially inactivating mutations “hypomorphs.” One of these hypomorphs is a SNP that exists in 6%–10% of Africans and 1%–2% of African Americans, which changes proline at amino acid 47 to serine (Pro47Ser; P47S). We previously showed that the P47S variant of p53 is intrinsically impaired for tumor suppressor function, and that this SNP is associated with increased cancer risk in mice and humans. Here we show that this SNP also influences the tumor microenvironment, and the immune microenvironment profile in P47S mice is more protumorigenic. At basal levels, P47S mice show impaired memory T-cell formation and function, along with increased anti-inflammatory (so-called “M2”) macrophages. We show that in tumor-bearing P47S mice, there is an increase in immunosuppressive myeloid-derived suppressor cells and decreased numbers of activated dendritic cells, macrophages, and B cells, along with evidence for increased T-cell exhaustion in the tumor microenvironment. Finally, we show that P47S mice demonstrate an incomplete response to anti-PD-L1 therapy. Our combined data suggest that the African-centric P47S variant leads to both intrinsic and extrinsic defects in tumor suppression. Significance: Findings presented here show that the P47S variant of TP53 influences the immune microenvironment, and the immune response to cancer. This is the first time that a naturally occurring genetic variant of TP53 has been shown to negatively impact the immune microenvironment and the response to immunotherapy.


Introduction
The tumor suppressor p53 plays a critical role in the cellular response to nearly all forms of stress, including oncogenic, genotoxic, and metabolic stress (1)(2)(3). In response to such stress, p53 becomes posttranslationally stabilized and activated, and subsequently regulates several tumor suppressive pathways that to double-strand breaks in DNA (23). The P47S variant exists in approximately 6%-10% of Africans and 1%-2% of African Americans, so studies in this area have the potential to aid in the understanding of cancer risk disparities in this population. In addition, whereas our group discovered that P47S tumors are generally poorly responsive to genotoxic stress, we were able to show that these tumors possess enhanced sensitivity to agents like BRD2/4 inhibitors and cisplatin (24), thus establishing the paradigm that one can tailor chemotherapy to genetic variants of TP.
There is emerging evidence that p53 plays a role in the regulation of the immune microenvironment (25,26). For example, wild-type (WT) p53 can stimulate the M1, or proinflammatory, polarization of macrophages (27). Stabilization of WT p53 can also lead to increased tumor infiltration of T lymphocytes and tumorsuppressing immune activity (28). Conversely, mutant p53 can stimulate M2, or anti-inflammatory, polarization of macrophages, thereby contributing to immunosuppression (29,30). For these reasons, and because of evidence for ethnic disparities in the response to immune checkpoint inhibitors (31,32), we sought to determine the impact of the P47S variant on the tumor microenvironment (TME).

Cell Lines and Culture Conditions
Murine colon cancer cells derived from a female animal, MC38 (RRID: CVCL_B288), were obtained from the ATCC and grown in McCoy's 5A medium (Gibco) supplemented with 10% FBS (HyClone, GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Corning Cellgro). Cells were grown in a 5% CO 2 humidified incubator at 37°C. Cell line validation was completed using short tandem repeat profiling. Cells were tested for Mycoplasma contamination 1 week prior to use.

Tumor Studies
All animal studies were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH. All protocols were approved by The Wistar Institute Institutional Animal Care and Use Committee. Hupki mice in a pure C57Bl/6 strain background [described previously (16)], homozygous for P47 (WT) or S47 TP, between 6 and 10 weeks of age were used for all experiments. In most cases, the mice analyzed are littermate mice from P47/S47 heterozygous crosses; in all cases, P47 and S47 mice were no more than two generations removed from a backcross to each other. Mice were housed in plastic cages with ad libitum diet and maintained with a 12-hour dark/12-hour light cycle at 22°C. For tumor engraftment experiments, 1 × 10 6 MC38 cells were injected subcutaneously into the right flanks of 6-10 weeks old male mice. For CD8 + T-cell depletion experiments, mice were treated with 200 μg anti-CD8b antibody (anti-mouse CD8b BioXCell, catalog no. BE0223) via intraperitoneal injection on the following schedule: 1 week prior to MC38 injections, 2 days prior to MC38 injections, two times per week for the duration of the study. For PD-L1 blockade therapy experiments, mice were administered three doses of anti-CD274 antibody (B7-H1, PD-L1; BioLegend: 124339) or three doses of rat IgG2b isotype control antibody (BioLegend: 400672) by intraperitoneal injection on the following schedule: 7 days post-MC38 injection, 10 days post-MC38 injection, 13 days post-MC38 injection. Tumor volumes were measured using digital calipers and tumor volume was calculated using the formula: volume = (length × width 2 ) × 0.52. All mice were monitored daily for signs of pain or distress. Tumor volumes were measured every other day until the study endpoint, at which point all mice were euthanized.

Statistical Analysis
All statistical analyses between two groups were performed using the Wilcoxon rank-sum test when group sizes were small (<6 datapoints) or the Student t test when sample sizes were larger (e.g., Fig. 2

Data Availability
The data generated in this study are available upon request from the corresponding authors.

S47 Exhibits Poorer Extrinsic Tumor Suppressor Function, Compared with Wt P53
We previously showed that the P47S variant of p53 (hereafter denoted S47) shows an intrinsic defect in tumor suppressor function and is associated with increased cancer risk (16,24). In the current study, we sought to probe the impact of the P47S variant on the TME. To do this, the MC38 murine colorectal tumor cell line was injected subcutaneously into the flanks of mice of identical age and sex containing WT p53 (hereafter P47) or the S47 variant, and tumor growth was assessed over time (Fig. 1A). In two independent studies, we found a significant increase in MC38 tumor growth in S47 hosts, suggesting an extrinsic tumor suppressor defect for this variant ( Fig. 1B and C). To distinguish whether this extrinsic defect was due to differences in adaptive immune function or other components of the TME, we performed a similar study and included experimental groups where both genotypes of mice were depleted for CD8 + T cells via administration of an anti-CD8b antibody. In the control groups, tumors again grew significantly larger in S47 hosts (Fig. 1D); however, this observed difference in tumor growth between genotypes was abolished when CD8 + T cells were depleted (Fig. 1E). Successful CD8 + T-cell depletion in the tissues was confirmed by flow cytometry (Fig. 1F). These data suggest that an altered immune microenvironment may underlie an extrinsic tumor suppressor defect in S47 mice.

Basal Differences in the Level and Function of T Cells from S47 Mice
We next sought to assess whether P47 and S47 mice possessed basal differences in immune cell phenotype and function. Toward this goal, we analyzed non-TBM of identical age and sex for the level of different subsets of immune cells. Because CD8 + T-cell depletion abolished the differences in tumor growth, and because we previously reported that S47 mice show increased populations of M2-like (anti-inflammatory) macrophages (35), we focused on these populations of immune cells. Our flow cytometric analyses revealed a significantly reduced population of memory (CD44 + ) CD4 + T cells in S47 mice ( Fig. 2A). Specifically, we observed a significant reduction in central memory (CD44 + and CD62L + ) CD4 + T cells in S47 mice (Fig. 2B). Conversely, we noted an increase in certain polarized mature CD4 + T helper subsets as evidenced by significantly elevated frequencies of CX 3 CR1 + and CXCR5 + CD4 + T cells in S47 mice ( Fig. 2C and D), which are associated with Th1-polarized and T follicular helper cells, respectively. We found no evidence for differences in total memory CD8 + T cells between P47 and S47 mice (Fig. 2E); however, like the CD4 + T-cell compartment, we observed a significant reduction in CD8 + central memory T cells in S47 mice (Fig. 2F). Finally, we found evidence for a significant increase in activated CD8 + effector T cells in S47 mice, as evidenced by an increased frequency of %CXCR3 + CD8 + T cells (Fig. 2G). Our combined data are suggestive of a deficit in central memory T cell formation in S47 mice, accompanied by an increase in differentiated T helper and T effector subsets.
We next assessed basal T-cell effector function in P47 and S47 mice. Upon stimulation with Concanavalin A (ConA), we found a significantly reduced frequency of degranulating (CD107a + ) CD4 + and CD8 + T cells in S47 mice (Fig. 3A). These analyses also revealed a significant deficit in the production of effector cytokines, such as IFNγ and TNFα, from S47 CD8 + T cells, specifically in response to stimulation by anti-CD3/CD28 beads and PMA/Ionomycin ( Fig. 3B and C). We next assessed polyfunctionality profiles of T cells derived from S47 and P47 mice after stimulation. We observed a significant reduction in CD107a + /TNFα + T cells from S47 mice when stimulated with ConA or PMA/Ionomycin ( Fig. 3D and F), and a reduction in CD107a + /Perforin + /TNFα + T cells in S47 mice when stimulated with anti-CD3/CD28 beads (Fig. 3E). These combined data support the premise that T cells from S47 mice show reduced effector function following stimulation.

FIGURE 1
The tumor growth advantage in S47 mice is abolished following CD8 + T-cell depletion. A, Illustration of the experimental approach.
Replicate P47 and S47 mice were injected subcutaneously with 1 × 10 6 MC38 tumor cells. At day 19, spleens and tumors from mice were harvested and analyzed by flow cytometry. B, Tumor growth curves for P47 and S47 mice injected with MC38 cells. n = 5 mice per group. Linear mixed model used for statistical analysis. **, P < 0.01. C, Tumor weights of MC38 tumors harvested from P47 and S47 mice. n = 5 mice per group. Wilcoxon rank-sum test used for statistical analysis. *, P < 0.05. D and E, Tumor growth curves for P47 and S47 mice injected with MC38 cells, ± treatment with anti-CD8b antibody. n = 5 mice per group. Linear mixed model used for statistical analysis. *, P < 0.05. F, The percentage of CD8 + T cells of total T cells (CD45 + /CD3 + ) in P47 and S47 mice before and after CD8 + T-cell depletion is shown on the left. Shown on the right are the total counts of splenic CD8 + T cells in P47 and S47 mice before and after CD8 + T-cell depletion.
To extend these studies, we next analyzed macrophage function in P47 and S47 mice, by purifying BMDMs. We found evidence that upon stimulation, S47 BMDM exhibit lower levels of proinflammatory cytokines, IL6 and IL12p40, and higher levels of the anti-inflammatory cytokine IL10 (Supplementary Fig. S1A), along with an increase in ARG expression ( Supplementary   Fig. S1B). These data are consistent with findings from our previous study, which indicated that S47 macrophages appear to be polarized toward an M2 immunosuppressive phenotype (35). Our combined data suggest the possibility of a more protumor immune microenvironment in S47 mice, evidenced from a basal decrease in S47 cytotoxic T-cell function following

Increased Immunosuppressive Immune Cells in the Spleens of Tumor-bearing S47 Mice
It was next logical to identify any alterations in the immune systems of S47 TBM compared with TBM of equal age and sex with WT p53 (P47). To address this, we analyzed tumors and splenocytes from S47 and P47 TBM for immune cell distribution and activation phenotype by flow cytometry. We found no evidence for differences in the levels of populations of immune cells in the tumors of P47 and S47 mice, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages, dendritic cells (DC), or CD4 + and CD8 + T cells ( Supplementary Fig. S2A-S2C). However, we noted significant differences in immune populations in the spleens of P47 and S47 mice. Specifically, we noted an increased population of MDSC in S47 TBM as determined by coexpression of the MDSC markers, Gr1 and CD11b, in CD45 + cells (Fig. 4A). In addition, the splenic macrophages from S47 TBM showed significantly lower levels of the activation markers MHC-I and CD86 (Fig. 4B). We also tested P47 and S47 splenocytes for the presence of activated DCs and B cells. These analyses demonstrated that S47 TBM possess significantly lower levels of activated DCs and B cells, compared with P47 mice (Fig. 4C and D). Taken together, these data are suggestive of a more immunosuppressive microenvironment in the spleens of S47 TBM, which could in turn serve to attenuate the tumor-specific adaptive cellular immune response.

Evidence for Increased T-cell Exhaustion in S47 Tumor-infiltrating Lymphocytes
Our next goal was to determine whether there were any changes in the function of immune cells in tumor-bearing P47 and S47 mice. As stated previously, we found no significant differences in the frequencies of infiltrating immune cell subsets in tumors from P47 and S47 mice, suggesting no differences in trafficking of immune cells into tumors ( Supplementary Fig. S2A-S2C). However, due to reduced levels of MHC class I and CD86 on antigen-presenting cells in S47 TBM, we interrogated whether the frequency of tumor-specific CD8 + T cells were significantly different between S47 and P47 TBM. Dextramer staining for CD8 + T cells specific for the KILTFDRL (KL8) neoantigen of MC38 tumor cells, located in the ribosomal protein L18 (36), demonstrated that there was no difference in the frequency of tumor-associated KL8-specific dextramer positive (KL8-dex + ) CD8 + T cells in the TME between S47 and P47 mice, but that, unexpectedly, S47 mice harbored significantly elevated frequencies of KL8-dex + CD8 + T cells in the spleen (Supplementary Fig. S2D). These latter data suggest that the CD8 + T-cell defect in S47 mice is unlikely to be due to differences in priming of tumor antigen-specific responses or to differences in trafficking of antigen-specific CD8 + T cells to the tumor.

Next, we evaluated whether tumor-infiltrating lymphocytes (TIL) found in S47
TBM demonstrated alterations in transcription factor profiles or IRs, which would be indicative of attenuated functional capacity, when compared with P47 mice. There was no difference in the percentage of proliferating TILs as evidenced by Ki67 expression, indicating that S47-derived TILs are not anergic and respond to antigen (Fig. 5A). However, we did observe significantly altered transcription factor profiles in S47 TILs, with a higher frequency of CD4 + and CD8 + TILs expressing Eomes and Tbet in S47 TBM (Fig. 5B). Tbet phosphorylation and function have been linked to increased activation of the mTOR pathway (37), and we have recently published that S47 mice exhibit increased mTOR activity, compared with P47 mice (21). Consistent with this premise, in conjunction with elevated Tbet expression, we observed greater frequencies of phospho-Ser2448 mTOR + TILs in S47 TBM compared with P47 TBM (Fig. 5C). We also found that S47 TBM exhibited significantly higher frequencies of CD8 + Foxp3 + TILs; a similar increase was not observed in the CD4 + T-cell compartment (Fig. 5D). Collectively, the elevated expression of Eomes, Tbet, and Foxp3 suggest an activated but potentially functionally-exhausted T-cell phenotype which may contribute to attenuated tumor clearance in S47 mice. Interestingly, the differences in transcription factor expression profiles between S47 and P47 TBM exhibited similar trends when analyzing KL8-dex +

AACRJournals.org
Cancer Res Commun; 3(7) July 2023 CD8 + T cells derived from the tumor (Fig. 5E). In KL8-Dex + CD8 + T cells derived from the spleen, transcription factor levels were globally reduced, with no significant difference between S47 and P47 mice (Fig. 5F), suggesting that differences in transcription factor profiles between S47 and P47 TILs are antigen-driven.
The expression of IRs in TILs is another major determinant of T-cell exhaustion (38). Therefore, we next analyzed the tumor-infiltrating populations of CD4 + and CD8 + T cells for expression of the IRs lymphocyte activating protein 3 (LAG3), programmed cell death protein 1 (PD1), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and T-cell immunoglobulin and mucin domain 3 (Tim3). We observed a significant increase in the frequency of LAG3 + , PD1 + , and TIGIT + CD4 + TILs derived from S47 TBM, and a similar profile in CD8 + TILs with the exception of TIGIT which was globally lower on CD8 + TILs compared with CD4 + TILs ( Fig. 6A and B). We next analyzed the coexpression pattern of IRs on CD4 + and CD8 + TILs between genotypes. TILs derived from S47 mice demonstrated an overall greater proportion of subpopulations that expressed more than one IR (blue/purple colors), and fewer subpopulations that expressed a single IR (red colors) when compared with P47 mice ( Fig. 6C and D). This IR coexpression phenotype was dominated by a LAG3 + /PD1 + population which was found in significantly higher frequencies in both CD4 + and CD8 + TILs from S47 mice ( Fig. 6C and D, dark and light blue bars). Notably, the greatest proportion of IR + TILs from S47 mice were triple positive for LAG3, PD1, and TIGIT or LAG3, PD1, and Tim3 in CD4 + and CD8 + TILs, respectively, and these populations were significantly enriched compared with P47 TBM (Fig. 6C and D, dark blue bars). These data support the premise that tumor-infiltrating T cells in S47 mice may be functionally impaired due to elevated levels of IR coexpression.

Diminished Response of S47 TBM to Anti-PD-L1 Therapy
With the alterations in the immune microenvironment of S47 mice in mind, we next evaluated the efficacy of immune checkpoint blockade between genotypes. Toward this goal, we again analyzed MC38 tumor growth, as this tumor is known to respond to anti-PD-L1 therapy (39). We injected MC38 cells subcutaneously into P47 and S47 mice; after 7 days, when tumors were approximately 50 mm 3 , mice were randomly separated into two groups: one group received an IgG2b isotype control antibody and the other group received anti-PD-L1 antibody. Consistent with our previous results, tumors grew larger over time in S47 hosts compared with P47 (Fig. 7A). Both P47 and S47 TBM treated with anti-PD-L1 demonstrated tumor growth reduction when compared with genotype-matched isotype-treated controls, and when the increased growth rate of tumors in S47 hosts was taken into account, the overall fold decrease in tumor growth in P47 and S47 mice appeared similar (Fig. 7A). Notably, however, 4 of 9 P47 mice (44%) treated with anti-PD-L1 showed a complete ( Fig. 7B-D). In contrast, all S47 mice had palpable tumors at the end of the study (0% complete response), indicative of a delayed or poorer response to this immune checkpoint blockade.

Discussion
Previous work from our lab and others has shown that the S47 variant of TP shows impaired "tumor-intrinsic" tumor suppressor function. This impaired tumor suppressor function has been attributed to reduced serine 46 phos-phorylation (15), which is required for full p53-mediated apoptosis, as well as reduced sensitivity to ferroptosis (16), increased mTOR activity (21), decreased activity in replication error restart (22), and decreased ability to bind directly to sites of DNA damage (23). Herein, we show that this variant is associated with impaired "tumor-extrinsic" tumor suppressor function, as it is also associated with a more protumorigenic immune microenvironment. In our baseline immunophenotyping experiments, we found a deficit in the formation of central memory CD4 + and CD8 + T cells, and an increase in polarized CD4 + Th cells in S47 mice. Furthermore, upon stimulation, both CD4 + and CD8 + AACRJournals.org Cancer Res Commun; 3(7) July 2023  Individual tumor growth curves for each P47 and S47 mouse treated with rat IgG2b isotype control antibody or anti-PD-L1 antibody following MC38 cell injections. Statistics were derived from Wilcoxon rank-sum test. *, P < 0.05.
T cells from S47 mice show lower levels of degranulation, cytotoxicity, and effector cytokine production. Our analysis of BMDMs also demonstrates an anti-inflammatory/immunosuppressive phenotype in S47 mice. In TBM, our analysis of immune cell populations in the spleen shows an increase in MD-SCs, along with decreased activation of macrophages, DCs, and B cells. Future studies are needed to more comprehensively analyze the differences in immune populations using single-cell RNA sequencing or other corroborative approaches.
With respect to tumor-specific T-cell responses, we did not observe reductions in the frequency of neoantigen-specific CD8 + T cells either in the spleens or tumors of S47 mice, suggesting that there are no overt deficits in priming or trafficking of S47 T cells specific for MC38-derived neoantigens. We note that the mismatch between the murine p53 expressed by the transferred MC38 tumor cells and the humanized p53 gene expressed by S47 and P47 mice in the Hupki background could lead to a significant murine p53-specific T-cell response, which was not measured in this study. However, given that a role for CD8 + T cells in suppressing MC38 tumor growth has been reported in C57Bl6 mice with fully murine p53 where a p53 mismatch is absent (40,41), and that we have identified substantial frequencies of neoantigen-specific TILs in both P47 and S47 mice, it is unlikely that differences in a putative anti-murine p53 T-cell response between S47 and P47 mice are the primary driver of the differences in T-cell phenotypes between the p53 genotypes in this study. Nonetheless, this remains a possibility. Finally, we show evidence for increased markers of CD4 + and CD8 + T-cell exhaustion in S47 mice. All or many of these differences are likely to contribute to the increased tumor growth in S47 hosts, but the con-tribution of each of these parameters to the altered rate of tumor growth and differing efficacy of immunotherapy in S47 mice is likely to be complex. There have been other studies that have analyzed the impact of p53 loss or mutation on immune cell responses. However, most of the data published to date focuses on the immunologic outcomes associated with p53 knockout or "gain of function" mutants of p53 as opposed to hypomorphs that marginally affect p53 function. When p53 is absent in T cells only, there is a significant increase in T-cell effector functions (42), whereas p53 knockout in tumor cells is associated with a more suppressive tumor microenvironment (43,44). This work is the first to show an impact of a naturally-occurring hypomorphic variant of p53 on the adaptive immune response.
Two issues are unresolved in this study. The first is whether the defects in immune cell function evident in our S47 mice also occur in humans. Interestingly, and consistent with our findings, increased markers of T-cell exhaustion have been reported in African-descent individuals (32). However, whether this defect is linked to the S47 variant remains to be determined. At present, genome-wide association studies (GWAS) have failed to identify the S47 variant as one that is associated with disease, most likely because of the low incidence of this variant and the paucity of sample numbers of African-descent individuals in GWAS databases. Whereas our previous case-control study found a significant association of P47S with premenopausal breast cancer, the effect was modest and therefore P47S is likely a low penetrance allele for breast cancer (17). Further studies evaluating P47S and the efficacy of immune checkpoint inhibitors in African-descent populations are needed, and we cannot rule out the possibility that the immune defects we see in mice may not be seen in humans.
AACRJournals.org Cancer Res Commun; 3(7) July 2023 A second issue unresolved in this study is the mechanism whereby the S47 SNP contributes to an altered immune microenvironment in mice. WT p53 has also been shown to activate immune-regulating genes such as TAP1 and CD80 (45,46), and PD-L1 indirectly through its role in cellular senescence (47); while we have not identified these genes as differentially regulated in P47 and S47 cells, we cannot rule out the possibility that these genes could have impacted our results. Another possibility involves mTOR, the master regulator of metabolism.
Our previous studies showed that, in mouse and human cells containing the S47 variant, there is decreased repression of the cystine transporter SLC7A11, leading to increased synthesis of the antioxidants glutathione and coenzyme A (19,20). We showed that this increase in glutathione directly regulates the interaction of mTOR with Rheb; consequently, human and mouse cells containing the S47 variant show increased mTOR activity (21). For this reason, and given the prominent role of mTOR in immune cell differentiation and function (48), we monitored mTOR function in T cells from P47 and S47 mice and found evidence for increased mTOR activity (phospho-mTOR) in CD4 + and CD8 + TILs from S47 mice (Fig. 5C). mTOR has been found to stimulate the differentiation of effector T cells (49)(50)(51), and it inhibits the formation of CD8 + memory T cells (52,53). Moreover, the increased expression of Tbet, a transcription factor controlling T-cell effector function, is also driven by mTOR activity (54). Overall, our data best support the conclusion that the efficacy of immunotherapy may be impacted by the S47 SNP, and that prolonged immunotherapy, and/or simultaneous targeting of multiple IRs, may be necessary for complete tumor regression in this population. Our study and our model serve as an important experimental platform upon which to test these hypotheses.